R.V. wurde von einem Roche Postdoctoral Fellowship (2014-2017) unterstützt.

<ol>
	<li>Blanchette, M., Daneman, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+and+maintenance+of+the+BBB.">Formation and maintenance of the BBB.</a> Mech Dev. 138, 8-16 (2015).</li><li>Chow, B. W., Gu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Molecular+Constituents+of+the+Blood-Brain+Barrier.">The Molecular Constituents of the Blood-Brain Barrier.</a> Trends Neurosci. 38 (10), 598-608 (2015).</li><li>Niewoehner, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+Brain+Penetration+and+Potency+of+a+Therapeutic+Antibody+Using+a+Monovalent+Molecular+Shuttle.">Increased Brain Penetration and Potency of a Therapeutic Antibody Using a Monovalent Molecular Shuttle.</a> Neuron. 81 (1), 49-60 (2014).</li><li>Yu, Y. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+bispecific+antibodies+cross+the+blood-brain+barrier+in+nonhuman+primates.">Therapeutic bispecific antibodies cross the blood-brain barrier in nonhuman primates.</a> Sci Transl Med. 6 (261), 154 (2014).</li><li>Preston, J. E., Joan Abbott, N., Begley, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcytosis+of+macromolecules+at+the+blood-brain+barrier.">Transcytosis of macromolecules at the blood-brain barrier.</a> Adv Pharmacol. 71, 147-163 (2014).</li><li>De Bock, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Into+rather+unexplored+terrain-transcellular+transport+across+the+blood-brain+barrier.">Into rather unexplored terrain-transcellular transport across the blood-brain barrier.</a> Glia. 64 (7), 1097-1123 (2016).</li><li>Bien-Ly, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transferrin+receptor+(TfR)+trafficking+determines+brain+uptake+of+TfR+antibody+affinity+variants.">Transferrin receptor (TfR) trafficking determines brain uptake of TfR antibody affinity variants.</a> J Exp Med. 211 (2), 233-244 (2014).</li><li>Sade, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+human+blood-brain+barrier+transcytosis+assay+reveals+antibody+transcytosis+influenced+by+pH-dependent+receptor+binding.">A human blood-brain barrier transcytosis assay reveals antibody transcytosis influenced by pH-dependent receptor binding.</a> PLoS One. 9 (4), 96340 (2014).</li><li>Tian, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LRP-1-mediated+intracellular+antibody+delivery+to+the+Central+Nervous+System.">LRP-1-mediated intracellular antibody delivery to the Central Nervous System.</a> Sci Rep. 5, 11990 (2015).</li><li>Hsu, J., Rappaport, J., Muro, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+binding,+uptake,+and+transport+of+ICAM-1-targeted+nanocarriers+across+endothelial+and+subendothelial+cell+components+of+the+blood-brain+barrier.">Specific binding, uptake, and transport of ICAM-1-targeted nanocarriers across endothelial and subendothelial cell components of the blood-brain barrier.</a> Pharm Res. 31 (7), 1855-1866 (2014).</li><li>Siupka, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bidirectional+apical-basal+traffic+of+the+cation-independent+mannose-6-phosphate+receptor+in+brain+endothelial+cells.">Bidirectional apical-basal traffic of the cation-independent mannose-6-phosphate receptor in brain endothelial cells.</a> J Cereb Blood Flow Metab. , (2017).</li><li>Zlokovic, B. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+saturable+mechanism+for+transport+of+immunoglobulin+G+across+the+blood-brain+barrier+of+the+guinea+pig.">A saturable mechanism for transport of immunoglobulin G across the blood-brain barrier of the guinea pig.</a> Exp Neurol. 107 (3), 263-270 (1990).</li><li>Deane, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IgG-assisted+age-dependent+clearance+of+Alzheimer's+amyloid+beta+peptide+by+the+blood-brain+barrier+neonatal+Fc+receptor.">IgG-assisted age-dependent clearance of Alzheimer's amyloid beta peptide by the blood-brain barrier neonatal Fc receptor.</a> J Neurosci. 25 (50), 11495-11503 (2005).</li><li>Cabezon, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serial+block-face+scanning+electron+microscopy+applied+to+study+the+trafficking+of+8D3-coated+gold+nanoparticles+at+the+blood-brain+barrier.">Serial block-face scanning electron microscopy applied to study the trafficking of 8D3-coated gold nanoparticles at the blood-brain barrier.</a> Histochem Cell Biol. , (2017).</li><li>Gage, G. J., Kipke, D. R., Shain, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole+animal+perfusion+fixation+for+rodents.">Whole animal perfusion fixation for rodents.</a> J Vis Exp. (65), (2012).</li><li>Collin, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+uptake+of+tau/pS422+antibody+and+reduced+progression+of+tau+pathology+in+a+mouse+model+of+Alzheimer's+disease.">Neuronal uptake of tau/pS422 antibody and reduced progression of tau pathology in a mouse model of Alzheimer's disease.</a> Brain. 137 (10), 2834-2846 (2014).</li><li>Villasenor, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trafficking+of+Endogenous+Immunoglobulins+by+Endothelial+Cells+at+the+Blood-Brain+Barrier.">Trafficking of Endogenous Immunoglobulins by Endothelial Cells at the Blood-Brain Barrier.</a> Sci Rep. 6, 25658 (2016).</li><li>Villasenor, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Region-specific+permeability+of+the+blood-brain+barrier+upon+pericyte+loss.">Region-specific permeability of the blood-brain barrier upon pericyte loss.</a> J Cereb Blood Flow Metab. , (2017).</li><li>Armulik, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pericytes+regulate+the+blood-brain+barrier.">Pericytes regulate the blood-brain barrier.</a> Nature. 468 (7323), 557-561 (2010).</li><li>Richardson, D. S., Lichtman, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clarifying+Tissue+Clearing.">Clarifying Tissue Clearing.</a> Cell. 162 (2), 246-257 (2015).</li><li>Sibarita, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Deconvolution Microscopy. 95, 201-243 (2005).</li><li>Shaw, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Comparison of Widefield/Deconvolution and Confocal Microscopy for Three-Dimensional Imaging. , 453-467 (2006).</li><li>Chenouard, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Objective+comparison+of+particle+tracking+methods.">Objective comparison of particle tracking methods.</a> Nat Methods. 11 (3), 281-289 (2014).</li><li>Foret, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+general+theoretical+framework+to+infer+endosomal+network+dynamics+from+quantitative+image+analysis.">A general theoretical framework to infer endosomal network dynamics from quantitative image analysis.</a> Curr Biol. 22 (15), 1381-1390 (2012).</li><li>Daneman, R., Zhou, L., Kebede, A. A., Barres, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pericytes+are+required+for+blood-brain+barrier+integrity+during+embryogenesis.">Pericytes are required for blood-brain barrier integrity during embryogenesis.</a> Nature. 468 (7323), 562-566 (2010).</li><li>Bell, R. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pericytes+control+key+neurovascular+functions+and+neuronal+phenotype+in+the+adult+brain+and+during+brain+aging.">Pericytes control key neurovascular functions and neuronal phenotype in the adult brain and during brain aging.</a> Neuron. 68 (3), 409-427 (2010).</li><li>Ben-Zvi, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mfsd2a+is+critical+for+the+formation+and+function+of+the+blood-brain+barrier.">Mfsd2a is critical for the formation and function of the blood-brain barrier.</a> Nature. 509 (7501), 507-511 (2014).</li><li>Stewart, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+vesicles+in+the+blood-brain+barrier:+are+they+related+to+permeability.">Endothelial vesicles in the blood-brain barrier: are they related to permeability.</a> Cell Mol Neurobiol. 20 (2), 149-163 (2000).</li><li>Follain, G., Mercier, L., Osmani, N., Harlepp, S., Goetz, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Seeing+is+believing+-+multi-scale+spatio-temporal+imaging+towards+in+vivo+cell+biology.">Seeing is believing - multi-scale spatio-temporal imaging towards in vivo cell biology.</a> J Cell Sci. 130 (1), 23-38 (2017).</li></ol>

Autoren sind unter Erwerbstätigkeit von Roche.

Die oben beschriebene Protokoll beschreibt die Vorbereitung der Gehirn frei schwebenden Abschnitte, immunofluorescent Färbung, Bildaufnahme und Analyseparameter für hochauflösende Mikroskopie der BBB. Diese Methode hat vor kurzem verwendet, um die Lokalisierung der Antikörper Lieferung Plattformen3, den Transport von endogenen IgG über die BBB-17und die Heterogenität der BBB auf Pericyte Verlust18zu untersuchen. Verschiedene Schritte im Protokoll können zur Anpassung an die spezifischen Ziel des Experiments geändert werden. Zunächst erleichtert die Verwendung von dicken (100 µm) Abschnitte ihrer Handhabung beim Verfahren Immunostaining und Montage. Es ermöglicht auch für 3D Rekonstruktion des Kapillarnetzes, die neurovaskuläre Einheit und für die Generation der Kapillare und NVU Querschnitte. Allerdings Eindringen von Antikörper innerhalb der Gewebeschnitte variieren und einige Antikörper Färbung auf die oberflächliche Schicht des Gewebes in der Nähe des Deckglases beschränkt werden kann. Das Protokoll kann geändert werden, indem man die Konzentration der Reinigungsmittel während der Permeabilisierung Schritt bzw. die Länge der die Permeabilisierung Schritt zur Verbesserung der Antikörper Eindringen in das Gewebe. Zweitens kann Bildqualität gefährdet sein, wenn Sie versuchen, Bilder tiefer in das Gewebe (in der Regel 20 bis 30 µm unterhalb der Oberfläche) durch Lichtstreuung sowie optische Aberrationen von Brechungsindex Missverhältnis zu erwerben. Um dieses Problem zu überwinden, können neue Methoden für Gewebe, clearing und aktive Antikörper eindringen20 mit diesem Protokoll zu Bild größere Mengen an Gewebe kombiniert werden. Drittens erfolgt Dekonvolution nach der Bildaufnahme, axiale Auflösung des Bildes zu verbessern. Die Wahl des blind Deconvolution Algorithmus verwendet in diesem Protokoll stützte sich auf (i) seine einfache Bedienung, da keine Vorausberechnung der Point-Spread Funktion benötigt wird, (Ii) seine Robustheit für die Verbesserung der Bild Qualität21und (Iii) das Fehlen von Artefakten auf mIgG intrazelluläre Strukturen nach der Implementierung. Abhängig von der intrazellulären Strukturen visualisiert in der Probe führen andere Dekonvolution Algorithmen höhere Bildqualität. Die folgenden Referenzen21,22 geben eine ausführliche Diskussion über die Vorteile und Grenzen der zusätzliche Algorithmen für Bild Dekonvolution. Schließlich erlaubt die Verwendung von einer Bild-Analyse-Software-Paket die Segmentierung der Kapillaren und intrazellulären Strukturen in drei Dimensionen. Klar Bildanalyse beschränkt sich nicht auf die Software, die im Protokoll und alternative Pakete, zum Beispiel die Referenz23ausführlich beschrieben, Segment Bilder genutzt werden. Die Eignung der verschiedenen Software-Programmen für die Analyse der intrazellulären Strukturen über die BBB sollte empirisch überprüft werden, durch die Beurteilung der Genauigkeit der Bild Segmentierung.
Da diese Methode auf festen Proben basiert, bietet es keine direkten Informationen über die Dynamik des Transcytosis über die BBB. Jedoch mit Zeitverlauf Experimente24, z. B. kombinierbar durch intravenös injizieren das Molekül des Interesses und seine Ansammlung innerhalb BECs zu verschiedenen Zeitpunkten nach der Injektion zu rekonstruieren, die Kinetik der Messung intrazellulären Transport. Der Vorteil dieses Ansatzes ist, dass es für die Analyse von tiefen Hirnregionen wie gezeigt in18, die derzeit für intravitalen live bildgebenden Methoden nicht zugänglich sind. Ein wichtiger Schritt während des Protokolls ist die sorgfältige Überwachung der Gewebe Fixierung. Befestigung mit 4 % PFA drastisch reduziert die Immunogenität des intrazellulären Organellen und des endogenen oder peripher verabreichte Immunglobuline17 (Abbildung 1 b). Eine Einschränkung dieses Protokolls ist die Anforderung der qualitativ hochwertige Antikörper (d.h.niedrige unspezifische Färbung, geringe Kreuzreaktivität) geeignet für Immunfluoreszenz. Sofern solche Reagenzien zur Verfügung stehen, kann das Verfahren angewendet werden, um die intrazelluläre Lokalisation jedes Proteins des Interesses zu untersuchen. Zum Beispiel wurde das Protokoll zur Lysosomen im Gehirn endothelial Zellen17identifizieren. Es sollte auch angemerkt werden, dass da dieses Protokoll auf konfokalen Mikroskopie beruht, die laterale Auflösung durch Beugung begrenzt wird und nicht werden Strukturen, die kleiner als etwa 175 bis 250 aufgelöst kann nm (Abbildung 2).
Frühere Studien haben detaillierte Analyse der zellulären Zusammensetzung der konfokalen Mikroskopie25,26mit neurovaskuläre Einheit durchgeführt. Untersuchung von intrazellulären Transport an der BBB hängt jedoch vor allem auf die Verwendung von Transmission Electron Microscopy19,27,28. Während diese Methode die höchste laterale Auflösung der intrazellulären Strukturen bietet, bleibt Elektronenmikroskopie eine anspruchsvolle Technik mit niedrigem Durchsatz. Darüber hinaus ist die Anzahl der verschiedenen molekularen Ziele, die durch EM visualisiert werden können sehr begrenzt. Dieses Protokoll bietet eine zugängliche Alternative zu intrazellulären Transport an der BBB zu untersuchen. Die komplette Prozedur aus Gehirn Sammlung, Bildanalyse, kann in 5 bis 6 Tagen durchgeführt werden. Wenn geeignete Antikörper vorhanden sind, ermöglicht Immunfluoreszenz simultane Detektion von mehreren Zelle Arten/Moleküle innerhalb der gleichen Probe. Außerdem könnte dieses Protokoll mit Höchstauflösung Mikroskopie-Techniken zur Überwindung der Grenzen in räumlicher Auflösung29kombiniert werden. Insgesamt ermöglicht das oben beschriebene Protokoll die Quantifizierung von Veränderungen in die intrazelluläre Lokalisation innerhalb der neurovaskuläre Einheit interessierender Proteine. Seine Anwendung für verschiedene genetische oder pharmakologische Störungen ermöglicht die intrazellulären Struktur und Transport-Funktionen des BBB in Vivozu untersuchen.

Die Blut - Hirn-Schranke (BBB) ist eine kontinuierliche zellulären Barriere bilden Astrozyten, Perizyten, Neuronen und Endothelzellen, die das zentrale Nervensystem (ZNS) aus dem Blutzirkulation1trennt. Die Regulierung der Transport über die BBB spielt eine entscheidende Rolle bei der Aufrechterhaltung der Homöostase des Gehirns und wird durch spezielle Eigenschaften des Gehirns Endothelzellen (BECs) vermittelt. Das Vorhandensein von enge interzelluläre Verbindungen zwischen BECs und eine niedrige basale Transcytosis begrenzen den parazellulär und intrazelluläre Transport von Blut übertragbare Molekülen, bzw.2. Vor kurzem hat Transcytosis Weg in BECs genutzt, um Lieferung von therapeutischen große Moleküle an das Gehirn3,4zu erhöhen. Jedoch wurden die Mechanismen der Transcytosis über die BBB vollständig charakterisierten5,6noch nicht.
Umfangreiche Arbeit geleistet in Vitro zu entziffern, die zellulären und molekularen Mechanismen intrazellulären Transport über BECs7,8,9,10, 11, aber solche Systeme nicht die komplexe Architektur und Physiologie des Referats neurovaskuläre (NVU) rekapitulieren. Auf der anderen Seite Studien in Vivo12,13 enthalten detaillierte quantitative Informationen zu Frachten über die BBB aber keine Einblicke in die intrazellulären Mechanismen des Verkehrs. Untersuchung der zellulären und intrazellulären Komponenten der NVU bleibt in Vivo und ex Vivo daher sehr anspruchsvoll14. Nur eine begrenzte Anzahl von Techniken sind zugänglich, subzelluläre Strukturen in den Zellen der NVU zu analysieren. Die meisten Studien verwenden Elektronenmikroskopie, aber diese Technik ist begrenzt durch die komplexe Protokolle erforderlich für die ordnungsgemäße Gewebe Vorbereitung und Probenbehandlung. Daher haben wir eine Methode basiert auf hochauflösenden konfokalen Mikroskopie, die die Verarbeitung von Gehirn-Proben, die Analyse und Quantifizierung der subzelluläre Kompartimente innerhalb der Zellen von der NVU erleichtern würde.
Hier beschreiben wir ein Protokoll, das nutzt Maus Gehirn frei schwebenden Abschnitte um quantitative Bildgebung des BBB und NVU auf zellulärer und subzellulärer Ebene durchzuführen. Wir haben getestet und validiert eine Anzahl von Antikörpern gegen Bild und NVU in drei Dimensionen zu rekonstruieren. Darüber hinaus ermöglicht dieses Protokolls Bildgebung mit der maximalen optischen Beugung begrenzte Auflösung des Organellen innerhalb Gehirn-Kapillaren. Zusammen mit Bildanalyse kann dieses Protokoll verwendet werden, um den intrazellulären Transport von Makromolekülen in der BBB unter verschiedenen experimentellen Bedingungen, zum Beispiel in Mausmodellen Krankheit der Neurodegeneration zu untersuchen.

<table style="border-collapse:collapse;width:665pt" width="886">
	<colgroup>
		<col style="width:223pt" width="297" />
		<col style="width:98pt" width="130" />
		<col style="width:93pt" width="124" />
		<col style="width:251pt" width="335" />
	</colgroup>
	<tbody>
		<tr height="105" style="height:78.75pt">
			<td class="xl22" height="105" style="width:223pt;height:78.75pt" width="297">Rat monoclonal antibody clone ER-MP12 against CD31/PECAM</td>
			<td class="xl18" style="width:98pt" width="130">Novus Biologicals</td>
			<td class="xl18" style="width:93pt" width="124">MCA2388</td>
			<td class="xl18" style="width:251pt" width="335">Labels Brain Endothelial Cells 
			Use at dilution 1:100 
			detect with donkey polyclonal anti-rat IgG (H+L) coupled with suitable AlexaFluor dye, used at dilution 1:400</td>
		</tr>
		<tr height="105" style="height:78.75pt">
			<td class="xl22" height="105" style="width:223pt;height:78.75pt" width="297">Rat monoclonal antibody against Podocalyxin</td>
			<td class="xl18" style="width:98pt" width="130">R&#38;D Systems</td>
			<td class="xl18" style="width:93pt" width="124">MAB1556</td>
			<td class="xl18" style="width:251pt" width="335">Labels Lumen of capillaries 
			Use at dilution 1:100 
			detect with donkey polyclonal anti-rat IgG (H+L) coupled with suitable AlexaFluor dye, used at dilution 1:400</td>
		</tr>
		<tr height="105" style="height:78.75pt">
			<td class="xl22" height="105" style="width:223pt;height:78.75pt" width="297">Rabbit polyclonal antibody against CollagenIV</td>
			<td class="xl18" style="width:98pt" width="130">Biotrend</td>
			<td class="xl18" style="width:93pt" width="124">BT21-5014-70</td>
			<td class="xl18" style="width:251pt" width="335">Labels Basement membrane of capillaries 
			Use at dilution 1:100 
			detect with donkey polyclonal anti-rabbit IgG (H+L) coupled with suitable AlexaFluor dye, used at dilution 1:400</td>
		</tr>
		<tr height="105" style="height:78.75pt">
			<td class="xl22" height="105" style="width:223pt;height:78.75pt" width="297">Goat polyclonal antibody against CD13</td>
			<td class="xl18" style="width:98pt" width="130">R&#38;D Systems</td>
			<td class="xl18" style="width:93pt" width="124">AF2335</td>
			<td class="xl18" style="width:251pt" width="335">Labels pericytes 
			Use at dilution 1:100 
			detect with donkey polyclonal anti-goat IgG (H+L) coupled with suitable AlexaFluor dye, used at dilution 1:400</td>
		</tr>
		<tr height="42" style="height:31.5pt">
			<td class="xl22" height="42" style="width:223pt;height:31.5pt" width="297">Mouse monoclonal antibody clone G-A-5 against GFAP coupled to Cy3</td>
			<td class="xl18" style="width:98pt" width="130">Abcam</td>
			<td class="xl18" style="width:93pt" width="124">ab49874</td>
			<td class="xl18" style="width:251pt" width="335">Labels Astrocytes 
			Use at dilution 1:100</td>
		</tr>
		<tr height="105" style="height:78.75pt">
			<td class="xl22" height="105" style="width:223pt;height:78.75pt" width="297">Rabbit polyclonal antibody against Iba-1</td>
			<td class="xl18" style="width:98pt" width="130">Wako</td>
			<td class="xl18" style="width:93pt" width="124">019-19741</td>
			<td class="xl18" style="width:251pt" width="335">Labels Microglia 
			Use at dilution 1:100 
			detect with donkey polyclonal anti-rat IgG (H+L) coupled with suitable AlexaFluor dye, used at dilution 1:400</td>
		</tr>
		<tr height="105" style="height:78.75pt">
			<td class="xl22" height="105" style="width:223pt;height:78.75pt" width="297">Rat monoclonal antibody ABL93 gainst LAMP2</td>
			<td class="xl18" style="width:98pt" width="130">Fitzgerald</td>
			<td class="xl18" style="width:93pt" width="124">10R-CD107BBMSP</td>
			<td class="xl18" style="width:251pt" width="335">Labels Lysosomes 
			Use at dilution 1:100 
			detect with donkey polyclonal anti-rat IgG (H+L) coupled with suitable AlexaFluor dye, used at dilution 1:400</td>
		</tr>
		<tr height="42" style="height:31.5pt">
			<td class="xl23" height="42" style="width:223pt;height:31.5pt" width="297">donkey polyclonal antibody against mouse IgG (H+L) AlexaFluor594</td>
			<td class="xl18" style="width:98pt" width="130">LifeTechnologies</td>
			<td class="xl18" style="width:93pt" width="124">A21203</td>
			<td class="xl21">Use at dilution 1:400</td>
		</tr>
		<tr height="42" style="height:31.5pt">
			<td class="xl18" height="42" style="width:223pt;height:31.5pt" width="297">donkey polyclonal antibody against mouse IgG (H+L) AlexaFluor488</td>
			<td class="xl18" style="width:98pt" width="130">LifeTechnologies</td>
			<td class="xl18" style="width:93pt" width="124">A21202</td>
			<td class="xl21">Use at dilution 1:400</td>
		</tr>
		<tr height="42" style="height:31.5pt">
			<td class="xl18" height="42" style="width:223pt;height:31.5pt" width="297">goat polyclonal antibody against mouse IgG (H+L) AlexaFluor555</td>
			<td class="xl18" style="width:98pt" width="130">LifeTechnologies</td>
			<td class="xl18" style="width:93pt" width="124">A21422</td>
			<td class="xl21">Use at dilution 1:400</td>
		</tr>
		<tr height="42" style="height:31.5pt">
			<td class="xl18" height="42" style="width:223pt;height:31.5pt" width="297">Filter paper</td>
			<td class="xl18" style="width:98pt" width="130">Sigma-Aldrich</td>
			<td class="xl18" style="width:93pt" width="124">WHA10334347</td>
			<td class="xl18" style="width:251pt" width="335">Whatman prepleated qualitative filter paper 
			Grade 0858 1/2, grained</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl18" height="21" style="width:223pt;height:15.75pt" width="297">Cyanoacrylate glue</td>
			<td class="xl18" style="width:98pt" width="130">Roth Carl</td>
			<td align="right" class="xl18" style="width:93pt" width="124">258.1</td>
			<td class="xl18" style="width:251pt" width="335">Roti Coll 1</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl18" height="21" style="width:223pt;height:15.75pt" width="297">Fluorescent Mounting medium</td>
			<td class="xl18" style="width:98pt" width="130">Dako</td>
			<td class="xl18" style="width:93pt" width="124">S3023</td>
			<td class="xl18" style="width:251pt" width="335">Dako fluorescent mounting medium</td>
		</tr>
		<tr height="42" style="height:31.5pt">
			<td class="xl18" height="42" style="width:223pt;height:31.5pt" width="297">Adult mice</td>
			<td class="xl18" style="width:98pt" width="130">The Jackson Laboratory</td>
			<td class="xl18" style="width:93pt" width="124">000664</td>
			<td class="xl18" style="width:251pt" width="335">C57BL/6J male or female mice 
			between 9 and 19 months of age</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl18" height="21" style="width:223pt;height:15.75pt" width="297">Vibratome</td>
			<td class="xl18" style="width:98pt" width="130">Leica Biosystems</td>
			<td align="right" class="xl18" style="width:93pt" width="124">14047235612</td>
			<td class="xl21">Leica VT100S vibrating blade microtome</td>
		</tr>
		<tr height="42" style="height:31.5pt">
			<td class="xl18" height="42" style="width:223pt;height:31.5pt" width="297">Laser Scanning Confocal microscope</td>
			<td class="xl18" style="width:98pt" width="130">Leica Microsystems</td>
			<td class="xl18" style="width:93pt" width="124">NA</td>
			<td class="xl18" style="width:251pt" width="335">Leica TCS SP8 X with HyD detectors 
			and White light laser</td>
		</tr>
		<tr height="42" style="height:31.5pt">
			<td class="xl18" height="42" style="width:223pt;height:31.5pt" width="297">Image processing software</td>
			<td class="xl18" style="width:98pt" width="130">Leica Microsystems</td>
			<td class="xl18" style="width:93pt" width="124">NA</td>
			<td class="xl21">Leica Application Suite AF version 3.1.0 build 8587</td>
		</tr>
		<tr height="42" style="height:31.5pt">
			<td class="xl18" height="42" style="width:223pt;height:31.5pt" width="297">Image analysis software</td>
			<td class="xl18" style="width:98pt" width="130">Bitplane scientific software</td>
			<td class="xl18" style="width:93pt" width="124">NA</td>
			<td class="xl21">Imaris version 7.6.5 build 31770 for x64</td>
		</tr>
		<tr height="99" style="height:74.25pt">
			<td class="xl18" height="99" style="width:223pt;height:74.25pt" width="297">Fluorescence SpectraViewer</td>
			<td class="xl18" style="width:98pt" width="130">ThermoFisher Scientific</td>
			<td class="xl18" style="width:93pt" width="124">NA</td>
			<td class="xl25" style="width:251pt" width="335">https://www.thermofisher.com/ch/en/home/life-science 
			/cell-analysis/labeling-chemistry/fluorescence-spectraviewer.html</td>
		</tr>
	</tbody>
</table>

high-resolution confocal imaging of the blood-brain barrier: imaging, 3d reconstruction, and quantification of transcytosis

Die Blut - Hirn-Schranke (BBB) ist eine dynamische mehrzelligen Schnittstelle, die den Transport von Molekülen zwischen den Kreislauf und das Gehirn reguliert. Transcytosis über die BBB regelt die Lieferung von Hormonen, Metaboliten und therapeutische Antikörper, die in Anwesenheit von. Hier präsentieren wir eine Protokoll, die Immunfluoreszenz frei schwebenden Abschnitte mit Laser kombiniert-scanning-konfokalen Mikroskopie und Bildanalyse subzellularen Organellen innerhalb von Endothelzellen an der BBB zu visualisieren. Kombination dieser Datensatz mit 3D Bildanalyse-Software ermöglicht für die semi-automatischen Segmentierung und Quantifizierung der Kapillare Volumen und Fläche, sowie die Anzahl und Intensität von intrazellulären Organellen an der BBB. Die Erkennung der Maus endogenen Immunglobulin (IgG) in intrazellulären Vesikeln und deren Quantifizierung an der BBB wird verwendet, um die Methode zu veranschaulichen. Dieses Protokoll kann potenziell auf die Untersuchung der Mechanismen Steuern BBB Transcytosis von unterschiedlichen Molekülen in Vivoangewendet werden.

Repräsentative Beispiele von Bildern aus dem hier beschriebenen Protokoll abgerufen, Maus Gehirn Abschnitte waren voller Flecken mit Antikörpern, die verschiedene Komponenten der NVU, einschließlich die Basalmembran, die Astrozyten, die Perizyten zu erkennen sowie endothelialen Zellen (siehe Tabelle der Materialien für spezifische Antikörper verwendet) (Abbildung 1A, D und E). Bei dieser Auflösung ist es möglich, den astrocytic Einzelprozesse und Ende-Füße, die in direktem Kontakt mit Kapillaren sind zu unterscheiden.
Um die Eignung dieses Protokolls zur Erkennung von intrazellulären Strukturen hervorzuheben, wurden Gehirn Teile von Tieren, die am Rande mit den menschlichen Anti-Tau-mAb86 Antikörper16 injiziert mit einem Fluoreszent markierten Anti-Human-Antikörper ( gefärbt. Abbildung 1B). mAb86 ist speziell Ziel Neuronen mit dem Ausdruck einer pathologischen Form der Tau16bekannt. Hierin beschriebene Protokoll verwenden, wurde mAb86 mit Beugung begrenzte Auflösung innerhalb der einzelnen vesikuläre Strukturen innerhalb der Neuronen (Abbildung 1B-C) erkannt. Darüber hinaus war endogenen Maus IgG in intrazellulären Strukturen innerhalb der Endothelzellen, aber nicht in Perizyten (Abbildung 1-D-E und Abbildung 2) erkannt.
Der Erwerb von hochauflösenden konfokalen Z-Stapel des Gehirns Gefäßsystems ermöglicht dreidimensionale Segmentierung der Kapillaren und intrazellulären Vesikeln an der BBB. Abbildung 2 zeigt ein Beispiel für den Prozess der Rendern und Segmentierung eine Kapillare beschriftet mit CollagenIV und Maus IgG-positiven intrazellulären Vesikeln. Durch Quantifizierung der vollständigen Dataset segmentierte Bilder, zum Beispiel durch die Anzahl der Bläschen pro Kapillare Volumen messen, ist es möglich, Veränderungen in intrazelluläre Transportprozesse unter verschiedenen Bedingungen zu studieren. Abbildung 3 B-C zeigt die Unterschiede in mIgG Vesikel Anzahl und Fluoreszenz Intensität entsprechend mIgG bei der in Anwesenheit von, bzw. nach Erschöpfung der Pericyte im Mausmodell Pdgf-bret/ret als zuvor gemeldeten 17. der gleiche Ansatz wurde auch vor kurzem verwendet, um Änderungen in intrazellulären Transport an der BBB zwischen verschiedenen Regionen18zu analysieren.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56407/56407fig1.jpg"> 
Abbildung 1: Kennzeichnung von mehreren Zelltypen und subzelluläre Strukturen der neurovaskuläre Einheit Repräsentative Bilder der neurovaskuläre Einheit (A und D) und intrazellulären Vesikeln enthaltenen Neuronen (B) oder Endothelzellen (E) erhalten mit diesem Protokoll. Die maximale Intensität Projektionsbild in A (oben) zeigt die Verteilung der Astroglia-positiven Astrozyten (rot) umgebenden Kapillaren vom CollagenIV (grün) gekennzeichnet. Pfeile zeigen auf astrocytic Einzelprozesse. Maßstabsleiste = 20 µm. Bei dieser Auflösung sind die einzelnen Astrozyten Prozesse und Ende-Füße deutlich sichtbar, wie im gezoomten Bild die boxed Region (unten) dargestellt. Pfeilspitzen zeigen auf astrocytic Ende-Füße. Maßstabsleiste = 10 µm. Das Bild in B zeigt die Anhäufung von einem peripher injiziert Antikörper, mAb86 (grün), innerhalb einer hippocampal Neuronen. Pfeilspitzen zeigen, individuelle mAb86-positiven Vesikeln. Maßstabsleiste = 10 µm. Die Grafik in C zeigt die Linie Profil Intensität eines einzigen Vesikels. Die Vesikelgröße wurde aus die volle Breite am halben Maximum von einem "glockenförmig" Anfall (schwarze durchgezogene Linie) von der Intensitätskurve (grüne Linie und Kreis) geschätzt. Die Bilder in D zeigen eine dreidimensionale Rekonstruktion einer endothelial Zelle (grün), umgeben von einer Pericyte (rot) innerhalb der Basallamina (CollagenIV, grau). Die unteren Platten zeigen die einzelnen Fluoreszenz-Kanäle. Maßstabsleiste = 10 µm. Die Bilder in E zeigen die Lokalisierung von mIgG (rot) in intrazellulären Vesikeln innerhalb endothelial Zellen (CD31 in grün-Panel links) aber nicht im Perizyten (rechte Abbildung, CD13 in grün). In allen Bildern DAPI gefärbt Kerne sind in blau dargestellt. Maßstabsleiste = 5 µm. Platten D und E von Referenz17geändert wurden. <a href="//ecsource.jove.com/files/ftp_upload/56407/56407fig1large.jpg" target="_blank">Bitte klicken Sie hier für eine größere Version dieser Figur.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56407/56407fig2.jpg"> 
Abbildung 2 : Dreidimensionale Darstellung der Kapillaren und intrazellulären Vesikeln an der Blut - Hirn-Schranke. Mit dem beschriebenen Protokoll erwarb hochauflösende konfokale Bilder von CollagenIV-Positive Kapillaren (grün) und Maus IgG intrazelluläre Vesikeln (rot) wurden (A). Die linke Tafel zeigt einen optischen Bereich mit Querschnitten. Die Pfeile verweisen auf einzelne mIgG-positiven Vesikeln innerhalb Gehirn endothelial Zellen. Das Gremium für die rechts zeigt die 3D Rekonstruktion des Z-Fullstack mit image-Processing-Software (siehe Tabelle der Materialien). Die Kapillare Volumen (B) und einzelne Vesikel (C) wurden in drei Dimensionen gerendert und quantifiziert mit Bildbearbeitungssoftware (siehe Tabelle der Materialien). In allen Bildern DAPI gefärbt Kerne sind in blau dargestellt. Skalieren von Balken = 5 µm. <a href="//ecsource.jove.com/files/ftp_upload/56407/56407fig2large.jpg" target="_blank">Bitte klicken Sie hier für eine größere Version dieser Figur.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56407/56407fig3.jpg"> 
Abbildung 3 : Quantifizierung von mIgG intrazelluläre Lokalisation an der BBB. Repräsentative Bilder zeigen (A) dreidimensionale Rekonstruktionen der Kapillaren (beschriftet mit CollagenIV, grüne) und die Verteilung von mIgG (rot) bei C57BL/6 Mäusen und Pdgf-bret/ret Pericyte erschöpft Mäuse zuvor beschrieben im19. Skalieren von Balken = 10 µm. Die Bilder in B zeigen die Segmentierung von intrazellulären Vesikeln innerhalb der CollagenIV-Maske. The Graphen in B zeigt die Quantifizierung und Vergleich von mIgG Vesikel Anzahl pro Volumen der Kapillare. Jeder Punkt entspricht Messungen aus einzelnen Kapillaren Blöcken. Die durchgezogene Linie zeigt den Mittelwert und die Fehlerbalken repräsentieren die Standardabweichung der Daten. Die Bilder in C zeigen die mIgG Fluoreszenzsignal außerhalb der CollagenIV-Maske. Ebenso zeigt das Diagramm in C die Quantifizierung und Vergleich der Fluoreszenzintensität mIgG in das Gehirn Parenchym zwischen C57BL/6 Mäusen und Pdgf-bret/ret Pericyte erschöpft Mäuse. Fluoreszenz-Intensität-Einheiten wurden von der durchschnittlichen mIgG Parenchym Intensität gemessen an allen C57BL/6 Mäusen normalisiert. Diese Zahl wurde von Referenz17geändert. <a href="//ecsource.jove.com/files/ftp_upload/56407/56407fig3large.jpg" target="_blank">Bitte klicken Sie hier für eine größere Version dieser Figur.</a>

Watch this Scientific Journal Video about High-resolution Confocal Imaging of the Blood-brain Barrier: Imaging, 3D Reconstruction, and Quantification of Transcytosis at JoVE.com

High-resolution Confocal Imaging of the Blood-brain Barrier: Imaging, 3D Reconstruction, and Quantification of Transcytosis

Hochauflösende konfokale Bildgebung der Blut - Hirn-Schranke: Bildgebung, 3D-Rekonstruktion und Quantifizierung der Transcytosis

Hier präsentieren wir eine Mikroskopie-basiertes Protokoll für hochauflösende Bildgebung und eine dreidimensionale Rekonstruktion der Maus neurovaskuläre Einheit und Blut - Hirn-Schranke mit Gehirn frei schwebenden Abschnitten. Diese Methode ermöglicht die Visualisierung, Analyse und Quantifizierung der intrazellulären Organellen an der BBB.

The blood-brain barrier (BBB) is a dynamic multicellular interface that regulates the transport of molecules between the circulation and the brain. ...

high-resolution-confocal-imaging-blood-brain-barrier-imaging-3d

Research

JoVE Journal

Neuroscience

11.6K Aufrufe.  Roche Innovation Center Basel. Hier präsentieren wir eine Mikroskopie-basiertes Protokoll für hochauflösende Bildgebung und eine dreidimensionale Rekonstruktion der Maus neurovaskuläre Einheit und Blut - Hirn-Schranke mit Gehirn frei schwebenden Abschnitten. Diese Methode ermöglicht die Visualisierung, Analyse und Quantifizierung der intrazellulären Organellen an der BBB.

Serie: High-resolution Confocal Imaging of the Blood-brain Barrier: Imaging, 3D Reconstruction, and Quantification of Transcytosis

An Organotypic Slice Assay for High-Resolution Time-Lapse Imaging of Neuronal Migration in the Postnatal Brain

Neurogenesis in the postnatal brain depends on maintenance of three biological events: proliferation of progenitor cells, migration of neuroblasts, as well as differentiation and integration of new neurons into existing neural circuits. For postnatal neurogenesis in the olfactory bulbs, these events are segregated within three anatomically distinct domains: proliferation largely occurs in the subependymal zone (SEZ) of the lateral ventricles, migrating neuroblasts traverse through the rostral migratory stream (RMS), and new neurons differentiate and integrate within the olfactory bulbs (OB). The three domains serve as ideal platforms to study the cellular, molecular, and physiological mechanisms that regulate each of the biological events distinctly. This paper describes an organotypic slice assay optimized for postnatal brain tissue, in which the extracellular conditions closely mimic the in vivo environment for migrating neuroblasts. We show that our assay provides for uniform, oriented, and speedy movement of neuroblasts within the RMS. This assay will be highly suitable for the study of cell autonomous and non-autonomous regulation of neuronal migration by utilizing cross-transplantation approaches from mice on different genetic backgrounds.

This protocol describes an organotypic slice assay optimized for the postnatal brain and high-resolution time-lapse imaging of neuroblast migration in the rostral migratory stream.

Ein organotypischen Assay für High-Resolution Time-Lapse Imaging neuronaler Migration in der postnatalen Gehirn

Neurogenesis in the postnatal brain depends on maintenance of three biological events: proliferation of progenitor cells, migration of neuroblasts, as ...

Forschung

Neurowissenschaften

A Rapid Approach to High-Resolution Fluorescence Imaging in Semi-Thick Brain Slices

A fundamental goal to both basic and clinical neuroscience is to better understand the identities, molecular makeup, and patterns of connectivity that are characteristic to neurons in both normal and diseased brain. Towards this, a great deal of effort has been placed on building high-resolution neuroanatomical maps1-3. With the expansion of molecular genetics and advances in light microscopy has come the ability to query not only neuronal morphologies, but also the molecular and cellular makeup of individual neurons and their associated networks4. Major advances in the ability to mark and manipulate neurons through transgenic and gene targeting technologies in the rodent now allow investigators to 'program' neuronal subsets at will5-6. Arguably, one of the most influential contributions to contemporary neuroscience has been the discovery and cloning of genes encoding fluorescent proteins (FPs) in marine invertebrates7-8, alongside their subsequent engineering to yield an ever-expanding toolbox of vital reporters9. Exploiting cell type-specific promoter activity to drive targeted FP expression in discrete neuronal populations now affords neuroanatomical investigation with genetic precision.
Engineering FP expression in neurons has vastly improved our understanding of brain structure and function. However, imaging individual neurons and their associated networks in deep brain tissues, or in three dimensions, has remained a challenge. Due to high lipid content, nervous tissue is rather opaque and exhibits auto fluorescence. These inherent biophysical properties make it difficult to visualize and image fluorescently labelled neurons at high resolution using standard epifluorescent or confocal microscopy beyond depths of tens of microns. To circumvent this challenge investigators often employ serial thin-section imaging and reconstruction methods10, or 2-photon laser scanning microscopy11. Current drawbacks to these approaches are the associated labor-intensive tissue preparation, or cost-prohibitive instrumentation respectively.
Here, we present a relatively rapid and simple method to visualize fluorescently labelled cells in fixed semi-thick mouse brain slices by optical clearing and imaging. In the attached protocol we describe the methods of: 1) fixing brain tissue in situ via intracardial perfusion, 2) dissection and removal of whole brain, 3) stationary brain embedding in agarose, 4) precision semi-thick slice preparation using new vibratome instrumentation, 5) clearing brain tissue through a glycerol gradient, and 6) mounting on glass slides for light microscopy and z-stack reconstruction (Figure 1).
For preparing brain slices we implemented a relatively new piece of instrumentation called the 'Compresstome' VF-200 (http://www.precisionary.com/products_vf200.html). This instrument is a semi-automated microtome equipped with a motorized advance and blade vibration system with features similar in function to other vibratomes. Unlike other vibratomes, the tissue to be sliced is mounted in an agarose plug within a stainless steel cylinder. The tissue is extruded at desired thicknesses from the cylinder, and cut by the forward advancing vibrating blade. The agarose plug/cylinder system allows for reproducible tissue mounting, alignment, and precision cutting. In our hands, the 'Compresstome' yields high quality tissue slices for electrophysiology, immunohistochemistry, and direct fixed-tissue mounting and imaging. Combined with optical clearing, here we demonstrate the preparation of semi-thick fixed brain slices for high-resolution fluorescent imaging.

Here we describe a rapid and simple method to image fluorescently labeled cells in semi-thick brain slices. By fixing, slicing, and optically clearing brain tissue we describe how standard epifluorescent or confocal imaging can be used to visualize individual cells and neuronal networks within intact nervous tissue.

Eine schnelle Annäherung an High-Resolution Fluoreszenz-Imaging in Semi-Thick Hirnschnitten

A fundamental goal to both basic and clinical neuroscience is to better understand the identities, molecular makeup, and patterns of connectivity that are ...

Cerebrovascular Casting of the Adult Mouse for 3D Imaging and Morphological Analysis

Vascular imaging is crucial in the clinical diagnosis and management of cerebrovascular diseases, such as brain arteriovenous malformations (BAVMs). Animal models are necessary for studying the etiopathology and potential therapies of cerebrovascular diseases. Imaging the vasculature in large animals is relatively easy. However, developing vessel imaging methods of murine brain disease models is desirable due to the cost and availability of genetically-modified mouse lines. Imaging the murine cerebral vascular tree is a challenge. In humans and larger animals, the gold standard for assessing the angioarchitecture at the macrovascular (conductance) level is x-ray catheter contrast-based angiography, a method not suited for small rodents.
In this article, we present a method of cerebrovascular casting that produces a durable skeleton of the entire vascular bed, including arteries, veins, and capillaries that may be analyzed using many different modalities. Complete casting of the microvessels of the mouse cerebrovasculature can be difficult; however, these challenges are addressed in this step-by-step protocol. Through intracardial perfusion of the vascular casting material, all vessels of the body are casted. The brain can then be removed and clarified using the organic solvent methyl salicylate. Three dimensional imaging of the brain blood vessels can be visualized simply and inexpensively with any conventional brightfield microscope or dissecting microscope. The casted cerebrovasculature can also be imaged and quantified using micro-computed tomography (micro-CT)1. In addition, after being imaged, the casted brain can be embedded in paraffin for histological analysis.
The benefit of this vascular casting method as compared to other techniques is its broad adaptation to various analytic tools, including brightfield microscopic analysis, CT scanning due to the radiopaque characteristic of the material, as well as histological and immunohistochemical analysis. This efficient use of tissue can save animal usage and reduce costs. We have recently demonstrated application of this method to visualize the irregular blood vessels in a mouse model of adult BAVM at a microscopic level2, and provide additional images of the malformed vessels imaged by micro-CT scan. Although this method has drawbacks and may not be ideal for all types of analyses, it is a simple, practical technique that can be easily learned and widely applied to vascular casting of blood vessels throughout the body.

In this article, we present a simple, practical technique for cerebrovascular casting that is easy to perform and can be utilized to image the vascular tree of the adult mouse brain.

Zerebrovaskuläre Casting der erwachsenen Maus für 3D-Imaging und morphologische Analyse

Vascular imaging is crucial in the clinical diagnosis and management of cerebrovascular diseases, such as brain arteriovenous malformations (BAVMs). ...

High-resolution Functional Magnetic Resonance Imaging Methods for Human Midbrain

Functional MRI (fMRI) is a widely used tool for non-invasively measuring correlates of human brain activity. However, its use has mostly been focused upon measuring activity on the surface of cerebral cortex rather than in subcortical regions such as midbrain and brainstem. Subcortical fMRI must overcome two challenges: spatial resolution and physiological noise. Here we describe an optimized set of techniques developed to perform high-resolution fMRI in human SC, a structure on the dorsal surface of the midbrain; the methods can also be used to image other brainstem and subcortical structures.
High-resolution (1.2 mm voxels) fMRI of the SC requires a non-conventional approach. The desired spatial sampling is obtained using a multi-shot (interleaved) spiral acquisition1. Since, T2* of SC tissue is longer than in cortex, a correspondingly longer echo time (TE ~ 40 msec) is used to maximize functional contrast. To cover the full extent of the SC, 8-10 slices are obtained. For each session a structural anatomy with the same slice prescription as the fMRI is also obtained, which is used to align the functional data to a high-resolution reference volume.
In a separate session, for each subject, we create a high-resolution (0.7 mm sampling) reference volume using a T1-weighted sequence that gives good tissue contrast. In the reference volume, the midbrain region is segmented using the ITK-SNAP software application2. This segmentation is used to create a 3D surface representation of the midbrain that is both smooth and accurate3. The surface vertices and normals are used to create a map of depth from the midbrain surface within the tissue4.
Functional data is transformed into the coordinate system of the segmented reference volume. Depth associations of the voxels enable the averaging of fMRI time series data within specified depth ranges to improve signal quality. Data is rendered on the 3D surface for visualization.
In our lab we use this technique for measuring topographic maps of visual stimulation and covert and overt visual attention within the SC1. As an example, we demonstrate the topographic representation of polar angle to visual stimulation in SC.

This article describes techniques to perform high-resolution functional magnetic resonance imaging with 1.2 mm sampling in human midbrain and subcortical structures using a 3T scanner. Use of these techniques to resolve topographic maps of visual stimulation in the human superior colliculus (SC) is given as an example.

Hochauflösende Functional Magnetic Resonance Imaging Methods for Human Mittelhirn

Functional MRI (fMRI) is a widely used tool for non-invasively measuring correlates of human brain activity. However, its use has mostly been focused upon ...

High-resolution Live Imaging of Cell Behavior in the Developing Neuroepithelium

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central nervous system (CNS). Although much is known about the molecular mechanisms that pattern the spinal cord and elicit neuronal differentiation1, 2, we lack a deep understanding of these early events at the level of cell behavior. It is thus critical to study the behavior of neural progenitors in real time as they undergo neurogenesis.
In the past, real-time imaging of early embryonic tissue has been limited by cell/tissue viability in culture as well as the phototoxic effects of fluorescent imaging. Here we present a novel assay for imaging such tissue for long periods of time, utilizing a novel ex vivo slice culture protocol and wide-field fluorescence microscopy (Fig. 1). This approach achieves long-term time-lapse monitoring of chick embryonic spinal cord progenitor cells with high spatial and temporal resolution.
This assay may be modified to image a range of embryonic tissues3, 4 In addition to the observation of cellular and sub-cellular behaviors, the development of novel and highly sensitive reporters for gene activity (for example, Notch signaling5) makes this assay a powerful tool with which to understand how signaling regulates cell behavior during embryonic development.

Imaging embryonic tissue in real-time is challenging over long periods of time. Here we present an assay for monitoring cellular and sub-cellular changes in chick spinal cord for long periods with high spatial and temporal resolution. This technique can be adapted for other regions of the nervous system and developing embryo.

Hochauflösende Live-Bildgebung von Zell-Verhalten in der Entwicklung von Neuroepithel

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central ...

High-resolution In Vivo Manual Segmentation Protocol for Human Hippocampal Subfields Using 3T Magnetic Resonance Imaging

The human hippocampus has been broadly studied in the context of memory and normal brain function and its role in different neuropsychiatric disorders has been heavily studied. While many imaging studies treat the hippocampus as a single unitary neuroanatomical structure, it is, in fact, composed of several subfields that have a complex three-dimensional geometry. As such, it is known that these subfields perform specialized functions and are differentially affected through the course of different disease states. Magnetic resonance (MR) imaging can be used as a powerful tool to interrogate the morphology of the hippocampus and its subfields. Many groups use advanced imaging software and hardware (&#62;3T) to image the subfields; however this type of technology may not be readily available in most research and clinical imaging centers. To address this need, this manuscript provides a detailed step-by-step protocol for segmenting the full anterior-posterior length of the hippocampus and its subfields: cornu ammonis (CA) 1, CA2/CA3, CA4/dentate gyrus (DG), strata radiatum/lacunosum/moleculare (SR/SL/SM), and subiculum. This protocol has been applied to five subjects (3F, 2M; age 29-57, avg. 37). Protocol reliability is assessed by resegmenting either the right or left hippocampus of each subject and computing the overlap using the Dice&#39;s kappa metric. Mean Dice&#39;s kappa (range) across the five subjects are: whole hippocampus, 0.91 (0.90-0.92); CA1, 0.78 (0.77-0.79); CA2/CA3, 0.64 (0.56-0.73); CA4/dentate gyrus, 0.83 (0.81-0.85); strata radiatum/lacunosum/moleculare, 0.71 (0.68-0.73); and subiculum 0.75 (0.72-0.78). The segmentation protocol presented here provides other laboratories with a reliable method to study the hippocampus and hippocampal subfields in vivo using commonly available MR tools.

High-resolution In Vivo Manual Segmentation Protocol for Human Hippocampal Subfields Using 3T Magnetic Resonance Imaging

The goal of this manuscript is to study the hippocampus and hippocampal subfields using MRI. The manuscript describes a protocol for segmenting the hippocampus and five hippocampal substructures: cornu ammonis (CA) 1, CA2/CA3, CA4/dentate gyrus, strata radiatum/lacunosum/moleculare, and subiculum.

Hohe Auflösung<em&gt; In-vivo-</em&gt; Manuelle Segmentierung Protokoll für Menschen Hippocampus Teilfelder Verwenden von 3T Kernspintomographie

The human hippocampus has been broadly studied in the context of memory and normal brain function and its role in different neuropsychiatric disorders has ...

Ex Vivo Imaging of Postnatal Cerebellar Granule Cell Migration Using Confocal Macroscopy

During postnatal development, immature granule cells (excitatory interneurons) exhibit tangential migration in the external granular layer, and then radial migration in the molecular layer and the Purkinje cell layer to reach the internal granular layer of the cerebellar cortex. Default in migratory processes induces either cell death or misplacement of the neurons, leading to deficits in diverse cerebellar functions. Centripetal granule cell migration involves several mechanisms, such as chemotaxis and extracellular matrix degradation, to guide the cells towards their final position, but the factors that regulate cell migration in each cortical layer are only partially known. In our method, acute cerebellar slices are prepared from P10 rats, granule cells are labeled with a fluorescent cytoplasmic marker and tissues are cultured on membrane inserts from 4 to 10 hr before starting real-time monitoring of cell migration by confocal macroscopy at 37 &#176;C in the presence of CO2. During their migration in the different cortical layers of the cerebellum, granule cells can be exposed to neuropeptide agonists or antagonists, protease inhibitors, blockers of intracellular effectors or even toxic substances such as alcohol or methylmercury to investigate their possible role in the regulation of neuronal migration.

Ex Vivo Imaging of Postnatal Cerebellar Granule Cell Migration Using Confocal Macroscopy

During postnatal cerebellum development, immature granule cells originating from the germinal zone exhibit distinct modalities of migration to reach their final destination and to establish neuronal networks. This protocol describes the preparation of cerebellar slices and the confocal macroscopic approach used to investigate the factors that regulate neuronal migration.

Ex-vivo-Bildgebung von Postnatale Kleinhirn-Körnerzellmigration mittels konfokaler Makros

During postnatal development, immature granule cells (excitatory interneurons) exhibit tangential migration in the external granular layer, and then ...

High-resolution Structural Magnetic Resonance Imaging of the Human Subcortex In Vivo and Postmortem

The focus of this study was to test the resolution limits of structural MRI of a postmortem brain compared to living human brains. The resolution of structural MRI in vivo is ultimately limited by physiological noise, including pulsation, respiration and head movement. Although imaging hardware continues to improve, it is still difficult to resolve structures on the millimeter scale. For example, the primary visual sensory pathways synapse at the lateral geniculate nucleus (LGN), a visual relay and control nucleus in the thalamus that normally is organized into six interleaved monocular layers. Neuroimaging studies have not been able to reliably distinguish these layers due their small size that are less than 1 mm thick.
The resolving limit of structural MRI, in a postmortem brain was tested using multiple images averaged over a long duration (~24 h). The purpose was to test whether it was possible to resolve the individual layers of the LGN in the absence of physiological noise. A proton density (PD)1 weighted pulse sequence was used with varying resolution and other parameters to determine the minimum number of images necessary to be registered and averaged to reliably distinguish the LGN and other subcortical regions. The results were also compared to images acquired in living human brains. In vivo subjects were scanned in order to determine the additional effects of physiological noise on the minimum number of PD scans needed to differentiate subcortical structures, useful in clinical applications.

High-resolution Structural Magnetic Resonance Imaging of the Human Subcortex In Vivo and Postmortem

Here we present a protocol to determine the minimum number images that needed to be registered and averaged to resolve subcortical structures and test whether the individual layers of the LGN could be resolved in the absence of physiological noise.

Hochauflösende Strukturkernspintomographie des menschlichen Subcortex<I&gt; In-vivo-</I&gt; Und Postmortem

The focus of this study was to test the resolution limits of structural MRI of a postmortem brain compared to living human brains. The resolution of ...

High-Resolution Quantitative Immunogold Analysis of Membrane Receptors at Retinal Ribbon Synapses

Retinal ganglion cells (RGCs) receive excitatory glutamatergic input from bipolar cells. Synaptic excitation of RGCs is mediated postsynaptically by NMDA receptors (NMDARs) and AMPA receptors (AMPARs). Physiological data have indicated that glutamate receptors at RGCs are expressed not only in postsynaptic but also in perisynaptic or extrasynaptic membrane compartments. However, precise anatomical locations for glutamate receptors at RGC synapses have not been determined. Although a high-resolution quantitative analysis of glutamate receptors at central synapses is widely employed, this approach has had only limited success in the retina. We developed a postembedding immunogold method for analysis of membrane receptors, making it possible to estimate the number, density and variability of these receptors at retinal ribbon synapses. Here we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of retinal fixation, 2) freeze-substitution, 3) postembedding immunogold electron microscope (EM) immunocytochemistry and, 4) quantitative visualization of glutamate receptors at ribbon synapses.

The postembedding immunogold method is one of the most effective ways to provide high-resolution analyses of the subcellular localization of specific molecules. Here we describe a protocol to quantitatively analyze glutamate receptors at retinal ribbon synapses.

Hochauflösende Quantitative Immungold-Analyse von Membranrezeptoren auf der Netzhaut Ribbon Synapsen

Retinal ganglion cells (RGCs) receive excitatory glutamatergic input from bipolar cells. Synaptic excitation of RGCs is mediated postsynaptically by NMDA ...

Using a Microfluidics Device for Mechanical Stimulation and High Resolution Imaging of C. elegans

One central goal of mechanobiology is to understand the reciprocal effect of mechanical stress on proteins and cells. Despite its importance, the influence of mechanical stress on cellular function is still poorly understood. In part, this knowledge gap exists because few tools enable simultaneous deformation of tissue and cells, imaging of cellular activity in live animals, and efficient restriction of motility in otherwise highly mobile model organisms, such as the nematode Caenorhabditis elegans. The small size of C. elegans makes them an excellent match to microfluidics-based research devices, and solutions for immobilization have been presented using microfluidic devices. Although these devices allow for high-resolution imaging, the animal is fully encased in polydimethylsiloxane (PDMS) and glass, limiting physical access for delivery of mechanical force or electrophysiological recordings. Recently, we created a device that integrates pneumatic actuators with a trapping design that is compatible with high-resolution fluorescence microscopy. The actuation channel is separated from the worm-trapping channel by a thin PDMS diaphragm. This diaphragm is deflected into the side of a worm by applying pressure from an external source. The device can target individual mechanosensitive neurons. The activation of these neurons is imaged at high-resolution with genetically-encoded calcium indicators. This article presents the general method using C. elegans strains expressing calcium-sensitive activity indicator (GCaMP6s) in their touch receptor neurons (TRNs). The method, however, is not limited to TRNs nor to calcium sensors as a probe, but can be expanded to other mechanically-sensitive cells or sensors.

Using a Microfluidics Device for Mechanical Stimulation and High Resolution Imaging of C. elegans

New tools for mechanobiology research are needed to understand how mechanical stress activates biochemical pathways and elicits biological responses. Here, we showcase a new method for selective mechanical stimulation of immobilized animals with a microfluidic trap allowing high-resolution imaging of cellular responses.

Hochauflösende konfokale Bildgebung der Blut - Hirn-Schranke: Bildgebung, 3D-Rekonstruktion und Quantifizierung der Transcytosis

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